Hi January, To compare KO to WT, you need to do a separate channel anlaysis, using intraspotCorrelation() and lmscFit(). This will allow you to make any comparison you like, as if you had 40 single-channel arrays, but taking into account the two-colour structure. See the section on this in the limma User's Guide. Best wishes Gordon > Date: Mon, 17 Jan 2011 01:50:35 +0100 > From: January Weiner <january.weiner at="" mpiib-berlin.mpg.de=""> > To: BioC <bioconductor at="" stat.math.ethz.ch=""> > Subject: [BioC] Question about design in limma > Content-Type: text/plain; charset=ISO-8859-1 > > Hello, > > I've been presented with the following design: > > Tissue strain Cy5 Cy3 > 1 tis1 KO d0 d01 > 2 tis1 KO d01 d0 > 3 tis1 KO d0 d03 > 4 tis1 WT d03 d0 > 5 tis1 WT d0 d01 > 6 tis1 KO d03 d0 > 7 tis1 WT d01 d0 > 8 tis1 WT d0 d03 > 9 tis2 KO d0 d01 > 10 tis2 KO d01 d0 > 11 tis2 KO d0 d02 > 12 tis2 KO d02 d0 > 13 tis2 WT d0 d01 > 14 tis2 WT d01 d0 > 15 tis2 WT d0 d02 > 16 tis2 WT d02 d0 > 17 tis3 WT d0 d03 > 18 tis3 WT d03 d0 > 19 tis3 KO d03 d0 > 20 tis3 KO d0 d03 > > In summary: there are three different tissues. There are two strains > and four measurement points for the experiment (d0, d1, d2, d3), with > d0 as reference. The two-channel arrays have been loaded with RNA > samples such that the "day 0" from the respective tissue is the > reference. There is a dye swap present. > > I am looking for differences in the strains -- which genes are > regulated in a different manner between WT and KO. How should I do it > best? obviously, simply comparing WT with KO is *not* the way to go, > since change in d04 (as compared with d0) can be very different from > response in d02, and also response is very variable between tissues. > However, I am looking for genes that are, for example, differentially > expressed in the KO, but not changing in the WT. > > One way of solving that would be splitting the data sets, fitting the > model separately to the subsets, creating lists for subsets (e.g. list > of genes diff. expressed in KO, list of DE genes in WT) and comparing > the subsets. It works, but I don't like it. > > Best regards, > > j. > > -- > -------- Dr. January Weiner 3 -------------------------------------- > Max Planck Institute for Infection Biology > Charit?platz 1 > D-10117 Berlin, Germany > Web?? : www.mpiib-berlin.mpg.de > Tel? ?? : +49-30-28460514 ______________________________________________________________________ The information in this email is confidential and intend...{{dropped:4}}